Endothelins are peptides, that exist in three isoforms ET-1, ET-2, and ET-3, each encoded by a distinct gene. They have been originally discovered in the conditioned medium of porcine endothelial cells in 1988 by Yanagisawa (Yanagisawa M; Kurihara H; Kimura S; Tomobe Y; Kobayashi M; Mitsui Y; Yazaki Y; Goto K; Masaki T: A novel potent vasoconstrictor peptide produced by vascular endothelial cells [see comments]. NATURE (Mar. 31, 1988), 332(6163), 411-5.). The active ETs are peptides of 21 amino acids with two intramolecular disulfide bridges. They are produced from preproproteins of 203 to 212 amino acids which are processed by furin-like endopeptidases to the biologically inactive big-endothelin (big-ET). The big-ETs are specifically processed to mature ETs by a hydrolytic cleavage between amino acids 21 and 22 that are Trp21-Val22 (big-ET-1, big ET-2) and Trp21-Ile22 in big-ET-3 respectively. Already in 1988 a specific metalloprotease was postulated to be responsible for this specific cleavage. In 1994, ECE-1 (endothelin converting enzyme-1) was purified and cloned from bovine adrenal (Xu D, Emoto N, Giaid A, Slaughter C, Kaw S, de Witt D, Yanagisawa M: ECE-1: a membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1. Cell (1994) 78: 473-485).
ECE-1 is a membrane bound type II zinc-endopeptidase with a neutral pH optimum and a zinc binding motif HExxHx( greater than 20)E. It belongs to subfamily M13 and has a large 681 amino acid ectodomain that comprises the active site. Other members of the M13 family are NEP24.11 (neutral endopeptidase), PEX, a phosphate regulating neutral endopeptidase, and Kell blood group protein that has recently been described as a big-ET-3 processing enzyme. Members of the M13 family of human origin are characterized by a high molecular weight ( greater than 80 kDa) a number of conserved disulfide bridges and a complex glycosylation pattern. The structure of NEP has recently been solved. (Oefner et al, J. Mol. Biol. 2000, 296, 341-349). The catalytic domain of ECE and related human M13 proteinases are significantly larger ( greater than 650 amino acids) than members of matrix metalloproteases (MMPs). Unlike the family of the MMPs which belong to the metzincins and display a typical HExxHxxGxxH pattern members of the M13 family are gluzincins comprising a HExxHx( greater than 20)E pattern. These two families are clearly different in size of catalytic domains, structure and zinc coordinating pattern of ligands. Active sites of the two families show clear differences which has clear impact on type of inhibitors and the potential selectivity.
The present invention relates to compounds of formula (I) 
wherein
R1 is hydrogen, alkylcarbonyl or arylcarbonyl;
R2 is alkyl, alkinyl, hydroxyalkyl, carboxyalkyl, alkoxycarbonyl, alkylcarbonylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, alkylsulfonyl, aryl, arylalkyl, arylalkoxyalkyl, aryl(alkoxycarbonyl)alkyl, arylaminocarbonyl, diarylalkyl, aryl(carboxyalkyl) aminocarbonyl, arylcarbonyl, arylsulfonyl, cycloalkyl, cycloalkylcarbonyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, heterocyclylalkyl or the group YR2 is heterocyclyl;
R3 and R4 are independently selected from the group consisting of hydrogen, alkyl, alkylcycloalkyl, alkylcycloalkylalkyl, alkylthio, cycloalkyl, cycloalkylalkyl, carbamoyl, carboxy, carboxyalkyl, cyano, amino, mono- and dialkylamino, alkoxy, alkoxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, alkenyl, alkinyl, aryl, arylalkyl, arylalkyl(alkoxycarbonyl)alkyl, arylcarbonylalkyl, arylalkenyl, aryl(alkoxycarbonyl)alkyl, arylamino, arylalkylamino, aryloxy, halogen, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, trimethylsilanylethynyl or trifluormethyl;
R5 is hydrogen, alkyl, aryl, arylalkyloxycarbonyl, or alkylcarbonyl;
X1, X2, X3 and X4 are CH or N with the proviso that only up to two groups of X1, X2, X3 and X4 are N;
Y is xe2x80x94Oxe2x80x94 or xe2x80x94NR5xe2x80x94; and
dimeric forms, and/or pharmaceutically acceptable esters, and/or pharmaceutically acceptable salts thereof, preferably pharmaceutically acceptable esters, and/or pharmaceutically acceptable salts thereof, and most preferably pharmaceutically acceptable salts thereof.
The present invention is directed to compounds which are useful as inhibitors of metalloproteases, e.g. zinc proteases, particularly zinc hydrolases, and which are effective in the prophylaxis and treatment of disease states which are associated with vasoconstriction of increasing occurrences. Examples of such disorders are high blood pressure, coronary disorders, cardiac insufficiency, renal and myocardial ischaemia, renal insufficiency, dialysis, cerebral ischaemia, cardiac infarct, migraine, subarachnoid haemorrhage, Raynaud syndrome and pulmonary high pressure. In addition the compounds are useful as cytostatic and cerebroprotective agents for inhibition of graft rejection, for organ protection and for treatment of ophthalmological diseases.
The present invention is directed to compounds of formula (I): 
wherein
R1 is hydrogen, alkylcarbonyl or arylcarbonyl;
R2 is alkyl, alkinyl, hydroxyalkyl, carboxyalkyl, alkoxycarbonyl, alkylcarbonylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, alkylsulfonyl, aryl, arylalkyl, arylalkoxyalkyl, aryl(alkoxycarbonyl)alkyl, arylaminocarbonyl, diarylalkyl, aryl(carboxyalkyl)aminocarbonyl, arylcarbonyl, arylsulfonyl, cycloalkyl, cycloalkylcarbonyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, heterocyclylalkyl or the group YR2 is heterocyclyl;
R3 and R4 are independently selected from the group consisting of hydrogen, alkyl, alkylcycloalkyl, alkylcycloalkylalkyl, alkylthio, cycloalkyl, cycloalkylalkyl, carbamoyl, carboxy, carboxyalkyl, cyano, amino, mono- and dialkylamino, alkoxy, alkoxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, alkenyl, alkinyl, aryl, arylalkyl, arylalkyl(alkoxycarbonyl)alkyl, arylcarbonylalkyl, arylalkenyl, aryl(alkoxycarbonyl)alkyl, arylamino, arylalkylamino, aryloxy, halogen, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, trimethylsilanylethynyl or trifluormethyl;
R5 is hydrogen, alkyl, aryl, arylalkyloxycarbonyl, or alkylcarbonyl;
X1, X2, X3 and X4 are CH or N with the proviso that only up to two groups of X1, X2, x3 and X4 are N;
Y is xe2x80x94Oxe2x80x94 or xe2x80x94NR5xe2x80x94; and
dimeric forms, and/or pharmaceutically acceptable esters, and/or pharmaceutically acceptable salts thereof.
The term xe2x80x9calkylxe2x80x9d, alone or in combination, means a straight-chain or branched-chain alkyl group containing a maximum of 7, preferably a maximum of 4, carbon atoms, e.g., methyl, ethyl, n-propyl, 2-methylpropyl (iso-butyl), 1-methylethyl (iso-propyl), n-butyl, and 1,1-dimethylethyl (t-butyl).
The term xe2x80x9ccarboxyxe2x80x9d refers to the group xe2x80x94C(O)OH.
The term xe2x80x9ccarbamoylxe2x80x9d refers to the group xe2x80x94C(O)NH2.
The term xe2x80x9ccarbonylxe2x80x9d refers to the group xe2x80x94C(O)xe2x80x94.
The term xe2x80x9chalogenxe2x80x9d refers to the group fluoro, bromo, chloro and iodo.
The term xe2x80x9csulfonylxe2x80x9d refers to the group xe2x80x94S(O2)xe2x80x94.
The term xe2x80x9calkenylxe2x80x9d refers to a hydrocarbon chain as defined for alkyl having at least one olefinic double bond (including for example, vinyl, allyl and butenyl).
The term xe2x80x9calkinylxe2x80x9d refers to a hydrocarbon chain as defined for alkyl having at least one olefinic triple bond (including for example propinyl, butin-(1)-yl, etc.).
The term xe2x80x9calkoxyxe2x80x9d, alone or in combination, means an alkyl ether group in which the term xe2x80x98alkylxe2x80x99 has the significance given earlier, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec.butoxy, tert.butoxy and the like.
The term xe2x80x9calkoxycarbonylxe2x80x9d refers to a group of the formula xe2x80x94C(O)Rc wherein Rc is alkoxy as defined above.
The term xe2x80x9chydroxyxe2x80x9d refers to the group xe2x80x94OH, the term xe2x80x9ccyanoxe2x80x9d to the group xe2x80x94CN.
The term xe2x80x9chydroxyalkylxe2x80x9d means an alkyl group as defined above which is substituted by a hydroxy group.
The term xe2x80x9cthioalkylxe2x80x9d and xe2x80x9ccyanoalkylxe2x80x9d refer to an alkyl group as defined above which is substituted by a xe2x80x94SH group or an xe2x80x94CN group, respectively.
xe2x80x9cCarboxyalkylxe2x80x9d means a lower-alkyl as defined above which is substituted by a HOOCxe2x80x94 group.
The term xe2x80x9calkylcarbonylxe2x80x9d, alone or in combination, means an acyl group derived from an alkanecarboxylic acid, i.e. alkylxe2x80x94C(O)xe2x80x94, such as acetyl, propionyl, butyryl, valeryl, 4-methylvaleryl etc.
The term xe2x80x9ccycloalkylxe2x80x9d signifies a saturated, cyclic hydrocarbon group with 3-8, preferably 3-6 carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl and the like.
The term xe2x80x9caminoxe2x80x9d refers to the group xe2x80x94NH2.
The term xe2x80x9carylxe2x80x9d for R2xe2x80x94 alone or in combinationxe2x80x94, refers to an aromatic carbocyclic radical, i.e. a 6 or 10 membered aromatic or partially aromatic ring, e.g. phenyl, naphthyl or tetrahydronaphthyl, preferably phenyl or naphthyl, and most preferably phenyl. The aryl moiety is optionally substituted with one or more groups independently selected from halogen, preferably fluor, alkoxycarbonyl, e.g. methylcarbonyl, carboxy, cyano, alkyl, alkoxy, phenyl, phenoxy, trifluormethyl, trifluormethoxy, 1,3-dioxolyl, or 1,4-dioxolyl, more preferably fluor, alkoxycarbonyl, alkyl, trifluoromethyl and trifluoromethoxy and most preferably fluor. The most preferred aromatic groups are 2,5-difluorobenzyl and 2,4,5-trifluorobenzyl.
The term xe2x80x9carylxe2x80x9d for R3 and R4xe2x80x94alone or in combinationxe2x80x94, refers to an aromatic carbocyclic radical, i.e. a 6 or 10 membered aromatic or partially aromatic ring, e.g. phenyl, naphthyl or tetrahydronaphthyl, preferably phenyl or naphthyl, and most preferably phenyl. The aryl moiety is optionally substituted with one or more groups independently selected from halogen, alkoxycarbonyl, e.g. methylcarbonyl, carboxy, cyano, alkyl, alkoxy, phenyl, phenoxy, trifluormethyl, trifluormethoxy, 1,3-dioxolyl, or 1,4-dioxolyl, cyclohexyl, hydroxy, alkylamido, e.g. acetamido, nitro, alkylsulfonyl, e.g. methylsulfonyl, more preferably fluor, chlor, brom, alkoxy, carboxy, 1,4-dioxolyl, alkoxycarbonyl. The most preferred aromatic groups for R3 and R4 are phenyl and phenoxy.
The term xe2x80x9carylxe2x80x9d for R5 refers to phenyl optionally substituted with alkyl, alkoxy or halogen.
The term xe2x80x9caryloxyxe2x80x9d refers to an aryl group as defined above attached to a parent structure via an oxy radical, i.e., aryl-Oxe2x80x94.
The term xe2x80x9cheteroarylxe2x80x9d for R2xe2x80x94alone or in combinationxe2x80x94refers to an aromatic mono- or bicyclic radical having 5 to 10, preferably 5 to 6 ring atoms, containing one to three heteroatoms, preferably one heteroatom, e.g. independently selected from nitrogen, oxygen or sulfur. Examples of heteroaryl groups are thiophenyl, isoxazolyl, thiazolyl, pyridinyl, pyrrolyl, imidazolyl, tetrazolyl, preferably pyridinyl, isoxazolyl and thiazolyl. Optionally, the heteroaryl group can be mono-, di- or tri-substituted, independently, with phenyl, alkyl, alkylcarbonyl, alkoxycarbonyl, hydroxy, amino, alkylamino, dialkylamino, carboxy, alkoxycarbonylalkyl, preferably alkyl.
The term xe2x80x9cheteroarylxe2x80x9d for R3 and R4xe2x80x94alone or in combinationxe2x80x94refers to an aromatic mono- or bicyclic radical having 5 to 10, preferably 5 to 6 ring atoms, containing one to three heteroatoms, preferably one heteroatom, e.g. independently selected from nitrogen, oxygen or sulfur. Examples of heteroaryl groups are pyridinyl, thiophenyl, isoxyzolyl, isoquinolyl, quinolyl, and 1H-benzo[d][1,3]oxazin-2,4-dione and indolyl, pyrimidine, pyridazine, and pyrazine, preferably pyridinyl and thiophenyl. Optionally, the heteroaryl group can be mono, di- or tri-substituted, independently, with alkyl, alkoxy, halogen, alkylcarbonyl, alkoxycarbonyl, hydroxy, amino, alkylamino, dialkylamino, carboxy, alkoxycarbonylalkyl, preferably alkyl.
The term xe2x80x9cheterocyclylxe2x80x9d xe2x80x94alone or in combinationxe2x80x94refers to a non-aromatic mono- or bicyclic radical having 5 to 10, preferably 5 to 6 ring atoms, containing one to three heteroatoms, preferably one heteroatom, e.g. independently selected from nitrogen, oxygen or sulfur. Optionally the heterocyclic ring can be substituted by a group independently selected from halogen, alkyl, alkoxy, oxocarboxy, alkoxycarbonyl, etc. and/or on a secondary nitrogen atom (i.e. xe2x80x94NHxe2x80x94) by alkyl, arylalkoxycarbonyl, alkylcarbonyl or on a tertiary nitrogen atom (i.e. xe2x95x90Nxe2x80x94) by oxido. Examples for heterocyclic groups are morpholinyl, pyrrolidinyl, piperidyl, etc.
The term xe2x80x9cdimeric formxe2x80x9d means a compound wherein the two R1 groups of two identical compounds of formula I have been replaced by a common single bond or wherein R1 is glutathione-Sxe2x80x94 or cysteine-Sxe2x80x94 or ester and/or alkylcarbonyl or arylcarbonyl derivatives thereof, e.g. acetylcysteine-Sxe2x80x94 or benzoylcysteine-Sxe2x80x94, preferably glutathione-Sxe2x80x94, cysteine-Sxe2x80x94, acetylcysteine-Sxe2x80x94 or benzoylcysteine-Sxe2x80x94.
The term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d refers to those salts which retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable. The salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxylic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine and the like. In addition these salts may be prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from an inorganic base include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium salts and the like. Salts derived from organic bases include, but are not limited to salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polymine resins and the like.
xe2x80x9cPharmaceutically acceptable estersxe2x80x9d means that compounds of general formula (I) may be derivatised at functional groups to provide derivatives which are capable of conversion back to the parent compounds in vivo. Examples of such compounds include physiologically acceptable and metabolically labile ester derivatives, such as methoxymethyl esters, methylthiomethyl esters and pivaloyloxymethyl esters. Additionally, any physiologically acceptable equivalents of the compounds of general formula (I), similar to the metabolically labile esters, which are capable of producing the parent compounds of general formula (I) in vivo, are within the scope of this invention.
The compounds of formula (I) are useful in inhibiting mammalian metalloprotease activity, particularly zinc hydrolase activity. More specifically, the compounds of formula (I) are useful as medicaments for the treatment and prophylaxis of disorders which are associated with diseases caused by endothelin-converting enzyme (ECE) activity. Inhibiting of this enzyme would be useful for treating myocardial ischaemia, congestive heart failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma. In addition the compounds are useful as cytostatic and cerebroprotective agents, for inhibition of graft rejection, for organ protection and for treatment of ophthalmological diseases.
A preferred embodiment of the present invention encompasses compounds of general formula (II) 
wherein R1, R2, R3, R4, X1, X2, X3, X4 and Y are as defined in formula 1.
In a preferred embodiment of the invention R1 is selected from hydrogen or alkylcarbonyl, more preferably from hydrogen or acetyl and most preferably R1 is hydrogen.
In the above compounds R2 is preferably alkyl, alkinyl, hydroxyalkyl, carboxyalkyl, alkoxycarbonyl, alkylcarbonylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, alkylsulfonyl, aryl, arylalkyl, arylalkoxyalkyl, aryl(alkoxycarbonyl)alkyl, arylcarbamoyl, diarylalkyl, aryl(carboxyalkyl)amide, arylcarbonyl, arylsulfonyl, cycloalkyl, cycloalkylcarbonyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl or heterocyclylalkyl, more preferably R2 is aryl, arylalkyl, arylalkoxyalkyl, arylaminocarbonyl, arylcarbonyl, arylsulfonyl, cycloalkyl, cycloalkylcarbonyl, cycloalkylalkyl or heteroarylalkyl, even more preferably R2 is aryl, arylalkyl, arylcarbamoyl, arylamino, arylcarbonyl, arylsulfonyl or heteroarylalkyl and most preferably R2 is arylalkyl. In an especially preferred embodiment of the present invention R2 is phenylalkyl optionally substituted with 2 to 3 halogen atoms, preferably fluor atoms.
In the compounds of the present invention, preferably R3 and R4 are independently selected from the group consisting of hydrogen, alkyl, alkylthio, alkenyl, alkoxy, alkoxycarbonyl, amino, aryl, arylalkyl, arylalkenyl, arylalkylamino, aryloxy, mono- and dialkylamino, carbamoyl, carboxy, cyano, halogen, heteroaryl, heteroarylalkyl, trimethylsilanylethynyl and trifluoromethyl, more preferably R3 and R4 are independently selected from the group consisting of hydrogen, alkyl, alkoxy, alkoxycarbonyl, alkenyl, thiophenyl, amino, mono- and dialkylamino, carboxy, cyano, halogen, trimethylsilanylethynyl, phenylalkylamino, pyridinyl, pyrimidinyl, pyrazinyl, phenyl, and phenoxy, wherein the aryl and heteroaryl groups are optionally substituted with alkyl, alkoxy, carboxy, or halogen. In the most preferred embodiment of the present invention R3 is hydrogen, alkyl, alkoxy, alkoxycarbonyl, alkenyl, thiophenyl, amino, mono- and dialkylamino, carboxy, cyano, halogen, trimethylsilanylethynyl, phenylalkylamino, pyridinyl, pyrimidinyl, pyrazinyl, phenyl, and phenoxy, wherein the aryl and heteroaryl groups are optionally substituted with alkyl, alkoxy, carboxy, or halogen, and R4 is hydrogen.
In a further preferred embodiment of the present invention Y is xe2x80x94NR5xe2x80x94R5 being hydrogen or alkyl and more preferably hydrogen.
In another preferred embodiment of the present invention Y is xe2x80x94Oxe2x80x94.
The invention also relates to the above defined compounds wherein X1 is N and X2, X3 and X4 are CH, or wherein X2 is N and X1, X3 and X4 are CH or wherein X3 is N and X1, X2 and X4 are CH or wherein X1, X2, X3 and X4 are CH.
A preferred embodiment the present invention comprises compounds as defined above wherein R1 is hydrogen or alkylcarbonyl, R2 is phenylalkyl substituted with 2 to 3 halogen; R3 is selected from hydrogen, alkyl, alkoxy, alkoxycarbonyl, alkenyl, thiophenyl, amino, mono- and dialkylamino, carboxy, cyano, halogen, trimethylsilanylethynyl, phenylalkylamino, pyridinyl, pyrimidinyl, pyrazinyl, phenyl, or phenoxy, and wherein the aryl and heteroaryl groups are optionally substituted with alkyl, alkoxy, carboxy, or halogen; R4 is hydrogen; X1, X2, X3 and X4 are CH or N with the proviso that only up to two groups of X1, X2, X3 and X4 are N; and Y is xe2x80x94NHxe2x80x94 or xe2x80x94Oxe2x80x94. In a preferred embodiment R1 is hydrogen or acetyl and R2 is difluorobenzyl or trifluorobenzyl in the above defined compounds.
Preferred embodiments of the present invention are the compounds exemplified in the examples. Especially the present invention comprises compounds selected from the group consisting of
a) (3R,5S)-1-Pyrimidin-2-yl-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol trifluoro-acetate (1:1);
b) (3R,5S)-1-(4,6-Dimethoxy-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol;
c) (3R,5S)-1-(4-Amino-5-fluoro-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol;
d) 2-[(2S,4R)-4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]-nicotinonitrile;
e) (3R,5S)-1-(6-Phenyl-pyridazin-3-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol;
f) 2-[(2S,4R)-4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]-nicotinic acid;
g) 2-[(2S,4R)-4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]-6-methyl-pyrimidine-4-carboxylic acid methyl ester;
h) 2-[(2S,4R)-4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]-4-trifluoromethyl-pyrimidine-5-carboxylic acid methyl ester;
i) (3R,5S)-1-Pyrazin-2-yl-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol; compound with trifluoro-acetic acid;
j) 2-[(2S,4R)-4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]-nicotinamide;
k) (3R,5S)-5-(2,5-Difluoro-4-methoxy-benzyloxymethyl)-1-(2-methoxy-pyrimidin-4-yl)-pyrrolidine-3-thiol;
l) (3R,5S)-1-(2-Chloro-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol; compound with trifluoro-acetic acid;
m) (3R,5S)-1-(5-Ethyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol trifluoro-acetate (1:1);
n) (3R,5S)-1-(5-Propyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol trifluoro-acetate (1:1);
o) (3R,5S)-5-(2,4,5-Trifluoro-benzyloxymethyl)-1-(4-trifluoromethyl-pyrimidin-2-yl)-pyrrolidine-3-thiol trifluoro-acetate (1:1);
p) (3R,5S)-5-(2,4,5-Trifluoro-benzyloxymethyl)-1-(5-trifluoromethyl-pyridin-2-yl)-pyrrolidine-3-thiol trifluoro-acetate (1:1);
q) (3R,5S )-1-Pyridin-2-yl-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol trifluoro-acetate (1:1);
r) (2S,4R)-2-[4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]-6-methyl-pyrimidine-4-carboxylic acid;
s) (3R,5S)-1-(2-Methoxy-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol;
t) (3R,5S)-1-(2-Phenylamino-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol trifluoro-acetate (1:1);
u) (3R,5S)-1-(2-Benzylamino-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol; trifluoro-acetate (1:1);
v) (3R,5S)-1-(2-Methylamino-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol; trifluoro-acetate (1:1);
w) (3R,5S)-1-(2-Butylamino-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol; trifluoro-acetate (1:1);
x) (3R,5S)-1-(2-Methylsulfanyl-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol;
y) (3R,5S)-1-(2-Phenoxy-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol;
z) (3R,5S)-1-(5-Phenyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol;
aa) (3R,5S)-1-(5-Pyridin-2-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol; compound with trifluoro-acetic acid;
bb) (3R,5S)-1-(5-Pyridin-4-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol;
cc) (3R,5S)-1-(5-Thiophen-3-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol;
dd) (3R,5S)-1-[5-(4-Methoxy-phenyl)-pyrimidin-2-yl]-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol;
ee) (2S,4R)-4-{2-[4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]-pyrimidin-5-yl}-benzoic acid;
ff) (3R,5S)-1-(5-Allyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol;
gg) (3R,5S)-1-(5-Pyridin-3-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol; and
hh) (3R,5S)-5-(2,4,5-Trifluoro-benzyloxymethyl)-1-(5-trimethylsilanylethynyl-pyrimidin-2-yl)-pyrrolidine-3-thiol.
These compounds show IC50 values in the radioimmunoassay (E on ECE-inhibition, see blow) of about 0.5 nM to 100 nM.
Especially preferred compounds as defined by formula (I) are those selected from the group consisting of
a) (3R,5S)-1-Pyrimidin-2-yl-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol trifluoro-acetate (1:1);
b) (3R,5S)-1-(6-Phenyl-pyridazin-3-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol;
c) (3R,5S)-1-Pyrazin-2-yl-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol; compound with trifluoro-acetic acid;
d) (3R,5S)-1-(5-Ethyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol trifluoro-acetate (1:1);
e) (3R,5S)-1-(5-Propyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol trifluoro-acetate (1:1);
f) (3R,5S)-5-(2,4,5-Trifluoro-benzyloxymethyl)-1-(5-trifluoromethyl-pyridin-2-yl)-pyrrolidine-3-thiol trifluoro-acetate (1:1);
g) (3R,5S)-1-(5-Phenyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol;
h) (3R,5S)-Thioacetic acid S-[1-(5-propyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-3-yl]ester;
i) (3R,5S)-1-(5-Pyridin-2-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol; compound with trifluoro-acetic acid;
j) (3R,5S)-1-(5-Pyridin-4-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol;
k) 1-(5-Thiophen-3-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol;
l) 1-(5-Pyridin-3-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol;
m) (2S,4R)-5-[(2,5-Difluoro-benzylamino)-methyl]-1-(5-propyl-pyrimidin-2-yl)-pyrrolidine-3-thiol; and
n) (3R,5S)-Thioacetic acid S-[5-[(2,5-difluoro-benzylamino)-methyl]-1-(5-propyl-pyrimidin-2-yl)-pyrrolidin-3-yl]ester.
The invention also refers to pharmaceutical compositions containing a compound as defined above and a pharmaceutically acceptable excipient.
A further embodiment of the present invention refers to the use of compounds as defined above as active ingredients in the manufacture of medicaments comprising a compound as defined above for the prophylaxis and treatment of disorders which are caused by endothelin-converting enzyme (ECE) activity especially myocardial ischaemia, congestive heart failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma, graft rejection, diseases associated with cytostatic, ophthalmological, and cerebroprotective indications, and organ protection.
Further the invention refers to the use of compounds as described above for the treatment or prophylaxis of diseases which are associated with myocardial ischaemia, congestive heart failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma, graft rejection, diseases associated with cytostatic, ophthalmological, and cerebroprotective indications, and organ protection.
In addition the invention comprises compounds as described above for use as therapeutic active substances, in particular in context with diseases which are associated with zinc hydrolase activity such as myocardial ischaemia, congestive heart failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma, graft rejection, diseases associated with cytostatic, ophthalmological, and cerebroprotective indications, and organ protection.
The invention also comprises a method for the therapeutic and/or prophylactic treatment of myocardial ischaemia, congestive heart failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma, graft rejection, diseases associated with cytostatic, ophthalmological, and cerebroprotective indications, and organ protection, which method comprises administering a compound as defined above to a human being or animal.
The invention also relates to the use of compounds as defined above for the inhibition of zinc hydrolase activity.
The invention also refers to the above compounds whenever manufactured by a process as described below.
Compounds of formula (I) can be prepared by methods known in the art or as described below. Unless otherwise indicated, the substituents R1, R2, R3 R4, R5, X1, X2, X3,X4, and Y mentioned below are as defined above.
The process for the preparation of a compound as defined above may comprise the reaction of a compound of formula III 
wherein A is a HS- and P is a NH-protecting group as described in the following sections,
a) with a R2-halogenide for introduction of a xe2x80x94OR2 group followed by P-deprotection and introduction of a heteroaromate: or
b) first P-deprotection of formula (III), introduction of a heteroaromate as defined above followed by xe2x80x94OH/xe2x80x94NH2 replacement and reductive amination to introduce R2;
optionally followed by conversion of a R1, R2, R3, R4 group as above into a different one and/or deprotection and/or thiol liberation.
For the preparation of compounds of formula (I) the reaction pathway of scheme 1 can be followed: the starting material is commercial available or is synthesized from hydroxyproline by methods known in the art and described for example in xe2x80x9cThe Practice of Peptide Synthesisxe2x80x9d, M. Bodanszky and A. Bodanszky, Springer Verlag, Berlin, 1984.
The synthesis starts with the inversion of the configuration via preparation of the corresponding mesylate (e.g. reaction with MeSO3H/Ph3P/DIAD in toluene at RT to 80xc2x0 C.), via the chloride (e.g. reaction with Ph3P/CCl4 in CH2Cl2 at 3xc2x0 C. to RT) or via the bromide (e.g. reaction with LiBr/DEAD/Ph3P in THF at 4xc2x0 C. to RT). For retention of configuration the corresponding reaction may be performed with MeSO2Cl/pyridine/DMAP at 0xc2x0 C. to RT.
Step b of scheme 1 shows the introduction of the protected thio moiety, e.g. by reaction with triphenylmethanthiol or 4-methoxybenzylmercaptane (K-Ot-Bu in DMF for Cl: 0xc2x0 C., for Br: 0xc2x0 C. to RT, for Mes: RT to 100xc2x0 C.).
Reaction of step c of scheme 1 may be performed via Method A (LAH in THF at xe2x88x9220xc2x0 C.) or Method B (Red-Al in toluene/THF at xe2x88x9250xc2x0 C.).
Reaction of step d (for Yxe2x95x90xe2x80x94Oxe2x80x94) may be performed with
1. NaH/R2Br in DMF 0xc2x0 C. to RT, (O-alkylation)
2. TFA in CH2Cl2xe2x88x9220 to RT, (BOC deprotection)
3. Method A: 2-Chloro-hetero-aromate/N-ethyldiisopropylamine 3 h 80xc2x0 C., Method B (parallel-synthesis): 2-Chloro-hetero-aromate/N-ethyldiisopropylamine in dioxane or DMF, 16 h -2days 80-130xc2x0 C.,Method C (for less reactive compounds): 2-Chloro-hetero-aromate/N-ethyldiisopropylamine/CuI 10 h 80xc2x0 C.
For the preparation of phenolether compounds, the corresponding reaction may be performed under Mitsunobu conditions (DEAD/Ph3P/PhOH or PhSH in THF).
For Y being NR2or N-heterocycle a mesylation reaction may be performed: e.g.
1. 1.1 eq MeSO2Cl/1.5pyridine/1 eq DMAP, (mesylation);
2. YR2 is e.g. pyrrole, imidazol or, 1 eq NaI, NaH in DMF 0xc2x0 C. to RT;
3. iPr3SiH in TFA/CH2Cl2 or CH3CN (for trityl-thiol deprotection).
Thiol liberation may be performed with TFA/iPr3SiH in CH2Cl2 or CH3CN at RT.
An alternative route for the preparation of compounds with Y being N is: first P-deprotection (TFA in CH2Cl2 at xe2x88x9220xc2x0 C. to RT for Pxe2x95x90BOC), followed by reaction with 2-chloro-hetero-aromate/N-ethyldiisopropylamine/CuI for 10 h at 80xc2x0 C. (step f) followed by
1. phthalimide, DEAD/Ph3P in THF 0 to 80xc2x0 C., (phthalimide introduction under Mitsunobu condition)
2. hydrazine hydrate, EtOH, RT, (phthalimide deprotection) followed by reaction with the corresponding
3. aldehyde, SnCl2, NaBH3CN, MeOH, (reductive amination) (step g)
4. If necessary. R5 may be introduced by reaction with R5Br/K2CO3 in acetonitrile, RT, followed by reaction with
5. iPr3SiH in TFA/CH2Cl2 or CH3CN (for trityl-thiol deprotection). 
Scheme 2 summarizes special reaction pathways for the preparation of compounds of formula (I):
Scheme 2 A refers to a Cl-derivative as starting material which was synthesized as described in Scheme 1 (step e) with 2,4-dichloropyrimidine:
1. If XR3 is OMe the reaction may be performed with 3 eq MeOH/NaH in DMF, RT (4-fluoro-replacement takes place in YR2xe2x95x902,4,5-trifluoro benzyloxy ether derivatives).
2. If XR3 is OPh, the reaction may be performed with 10 eq PhOH/NaH in DMF for 8 h at 70xc2x0 C.
3. If XR3 is OMe, the reaction may be performed with 2.2 eq MeONa in MeOH at RT to 75xc2x0 C. (10 h) (no 4-fluoro-replacement takes place in YR2xe2x95x902,4,5-trifluoro-benzyloxy ether derivatives).
4. If XR3 is SMe, the reaction may be performed with 2.2 eq MeSNa/Nal in THF, RT to 70xc2x0 C. for 28 h.
5. If XR3 is NHR3, the reaction may be performed with 7.5-30 eq H2NR3/iPr2EtN in dioxane at 90-105xc2x0 C. for 48 h.
6. Thiol liberation may then be performed with TFA/iPr3SiH in CH2Cl2 or CH3CN at RT (step b).
The reaction pathway of scheme 2 B shows further synthesis routes for compounds of formula (I):
Reaction of step c) may be performed with the following methods:
1. Suzuki-coupling with ArylB(OH)2/Pd(PhP)4 in dimethoxyethane/EtOH and 2 M Na2CO3 2 h at 90xc2x0 C.; or
2. reaction with ArylB(OH)2/PdCl2(dppf) in dioxane and 2 M Na2CO3 for 24-48 h at 80xc2x0 C.; or
3. i. synthesis of a boron ester (e.g. 4,4,5,5-Tetramethyl-2-phenyl-[1,3,2]dioxaborolane derivative) by reaction with bis(pinacolato)diboron/KOAc/PdCl2(dppf) in DMF at 80xc2x0 C., and
4. ii. reaction with Bromoaromat/PdCl2(dppf)/2 M Na2CO3 for 16 h at 80xc2x0 C.; or Sonogashira-Hagihara coupling: reaction with ethinyltrimethylsilane/Et3N/PdCl2(Ph3P)2/CuI in DMF at 80xc2x0 C. 
Further derivatization of compounds of formula (I) is described in Scheme 3:
In case Y is nitrogen, reaction of step a may be performed with RCOCl, iPr2NEt, 4-(N-Benzyl-N-methylamino)pyridine polymer-supported, CH2Cl2 (N-acylation) followed by reaction with iPr3SiH, TFA, CH2Cl2, (thiol liberation). In case Y is protected nitrogen or oxygen, the reaction with the free thiol may be performed according to step c with RCOCl in pyridine at 0xc2x0 C. to RT or BOC-Cys(Npys)-OH (=2-(BOC-Cys)disulfanyl-3-nitro-pyridine) in DMF/0.1 M phosphate buffer (pH 6.2). In case of Y is a benzyloxy protected nitrogen, selective deprotection with 33% HBr in acetic acid at 0xc2x0 C. to RT is possible.
Dimeric forms of a compound of formula I may be prepared by oxidative treatment of the formula I monomers.
On the basis of their capability of inhibiting metalloprotease activity, especially zinc hydrolase activity, the compounds of formula I can be used as medicaments for the treatment and prophylaxis of disorders which are associated with vasoconstriction of increasing occurrences. Examples of such disorders are high blood pressure, coronary disorders, cardiac insufficiency, renal and myocardial ischaemia, renal insufficiency, dialysis, cerebral ischaemia, cardiac infarct, migraine, subarachnoid haemorrhage, Raynaud syndrome and pulmonary high pressure. They can also be used in atherosclerosis, the prevention of restenosis after balloon-induced vascular dilation, inflammations, gastric and duodenal ulcers, ulcus cruris, gram-negative sepsis, shock, glomerulonephtritis, renal colic, glaucoma, asthma, in the therapy and prophylaxis of diabetic complications and complications in the administration of cyclosporin, as well as other disorders associated with endothelin activities.
The ability of the compounds of formula (I) to inhibit metalloprotease activity, particularly zinc hydrolase activity, maybe demonstrated by a variety of in vitro and in vivo assays known to those of ordinary skill in the art. Pharmaceutically acceptable esters, pharmaceutically acceptable salts and dimeric forms of the compounds of formula I can also be tested by those of ordinary skill in the art for their ability to inhibit metalloprotease activity.
A) Cell Culture
A stable human umbilical vein endothelial cell line (ECV304) was cultured in xe2x80x9ccell factoriesxe2x80x9d as described until confluency (Schweizer et al. 1997, Biochem. J. 328: 871-878). At confluency cells were detached with a trypsin/EDTA solution and collected by low speed centrifugation. The cell pellet was washed once with phosphate buffered saline pH 7.0 and stored at xe2x88x9280xc2x0 C. until use.
B) Solubilization of ECE from ECV304 Cells
All procedures were performed at 0-4xc2x0 C. if not stated otherwise. The cell pellet of 1xc3x97109 cells was suspended in 50 ml of buffer A (20 mM Tris/HCl, pH 7.5 containing 5 mM MgCl2, 100 xcexcM PMSF, 20 xcexcM E64, 20 xcexcM leupeptin) and sonicated. The resulting cell homogenate was centrifuged at 100,000 gav for 60 minutes. The supernatant was discarded and the resulting membrane pellet was homogenized in 50 ml buffer A and centrifugated as described. The washing of the membrane fraction in buffer A was repeated twice. The final membrane preparation was homogenized in 50 ml of buffer B (buffer A+0.5% Tween 20 (v/v), 0.5% CHAPS (w/v), 0.5% Digitonin (w/v)) and stirred at 4xc2x0 C. for 2 hours. Thereafter the remaining membrane fragments were sedimented as described. The resulting clear supernatant containing the solubilized ECE was stored in 1.0 ml aliquots at xe2x88x92120xc2x0 C. until use.
C) ECE Assay
The assay measured the production of ET-1 from human big ET-1. To measure high numbers of samples an assay performed in 96 well plates was invented. The enzyme reaction and the radioimmunological detection of the produced ET-1 was performed in the same well, using a specifically developed and optimized coating technique.
D) Coating of Plates
Fluoronunc Maxisorp White (code 437796) 96 well plates were irradiated with 1 joule for 30 minutes in a UV Stratalinker 2400 (Stratagene). The 96 well plates were then fill with 300 xcexcl protein A solution (2 xcexcg/ml in 0.1 M Na2CO3 pH 9.5) per well and incubated for 48 hours at 4xc2x0 C. Coated plates can be stored for up to 3 weeks at 4xc2x0 C. until use.
Before use the protein A solution is discarded and the plates are blocked for 2 hours at 4xc2x0 C. with 0.5% BSA in 0.1M Na2CO3, pH 9.5.
Plates were washed with bidestilled water and were ready to perform the ECE assay.
E) Screening Assay
Test compounds are solved and diluted in DMSO. 10 xcexcl of DMSO was placed in the wells, followed by 125 xcexcl of assay buffer (50 mM Tris/HCl, pH 7.0, 1 xcexcM Thiorphan, 0,1% NaN3, 0.1% BSA) containing 200 ng big ET-1. The enzyme reaction was started by the addition of 50 xcexcl of solubilized ECE (diluted in assay buffer 1:30 to 1:60 fold (v/v)). The enzyme reaction was carried out for 30 minutes at 37xc2x0 C. The enzyme reaction was stopped by addition of 10 xcexcl 150 mM ETDA, pH 7.0.
Radioimmunoassay
The ET-1 RIA was performed principally as described earlier (Lxc3x6ffler, B.-M. and Maire, J.-P. 1994, Endothelium 1: 273-286). To plates containing the EDTA stopped enzyme reaction mixture 25 xcexcl of assay buffer containing 20000 cpm (3-(125I)Tyr)-endothelin-1 and 25 xcexcl of the ET specific antiserum AS-3 (dilution in assay buffer 1:1000) was added. Plates were incubated under mixing at 4xc2x0 C. over night. Thereafter, the liquid phase was sucked with a plate washer and plates were washed once with bidestilled water. To the washed plates 200 xcexcl scintillation cocktail (Microscint 40 LSC-Cocktail, Packard, code 6013641) was added and plates were counted for 2 minutes per well in a Topcount.
Standard curves were prepared in plates with synthetic ET-1 with final concentrations of 0 to 3000 pg ET-1 per well. In all plates controls for maximal ECE activity (in the presence of 10 xcexcl DMSO) and for background production of ET-1 immunoreactivity (in the presence of 10 mM EDTA or 100 xcexcM phosphoramidon) were performed. Assays were run in triplicate.
F) Kinetic Assay
The described assay format could be used to determine the kinetic characteristics of the used ECE preparation as well as different ECE inhibitors (i.e. Km, Ki) by variation of the substrate concentration used in the assay.
G) Cell based ECE Assay
Human ECE-1c was stable expressed in MDCK cells as described (Schweizer et al. 1997, Biochem. J. 328: 871-878). Cells were cultured in 24 well plates to confluency in Dulbecco""s modified Eagles""s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 0.8 mg/ml geneticin, 100 i.u./ml penicillin and 100 xcexcg/ml streptomycin in a humidified air/CO2 (19:1) atmosphere. Before ECE assay the medium was replaced by 0.5 ml DMEM-HBSS 1:1, 10 mM HEPES pH 7.0 supplemented with 0.1% (w/v) BSA. The inhibitors were added in DMSO at a final concentration of 1%. The enzyme reaction was started by the addition of 0.42 xcexcM human big ET-1 and performed for 1.5 hours at 37xc2x0 C. in an incubator. At the end of incubation, the incubation medium was quickly removed and aliquots were analysed by radioimmunoassay for produced ET-1 as described above.
The ECE screening assay was validated by the measurement of the characteristic inhibitor constants of phosphoramidon (IC50 0.8xc2x10.2 xcexcM) and CGS 314447 (IC50 20xc2x14 nM) [De Lombaert, Stephane; Stamford, Lisa B.; Blanchard, Louis; Tan, Jenny; Hoyer, Denton; Diefenbacher, Clive G.; Wei, Dongchu; Wallace, Eli M.; Moskal, Michael A.; et al. Potent non-peptidic dual inhibitors of endothelin-converting enzyme and neutral endopeptidase 24.11. Bioorg. Med. Chem. Lett. (1997), 7(8), 1059-1064]. The two inhibitors were measured with IC50 values not significantly different from those described in the literature but measured with different assay protocols. In the cell based assay phosphoramidon showed an IC50 of 4 xcexcM. This assay gave additional information about the inhibitory potency of inhibitors under much more physiologic conditions, as e.g. the ECE was embedded in a normal plasma membrane environment. It is important to state, that the screening assay was performed in the presence of 1 xcexcM Thiorphan to block any potential big ET-1 degradation due to the action of NEP24.11. No NEP activity was present in MDCK-ECE-1c transfected cells in preliminary experiments when ET-1 production was measured in presence or absence of thiorphan. In subsequent experiments no thiorphan was added in the incubation medium.
According to the above methods, the compounds of the present invention show IC50 values in the radioimmunoassay (E on ECE-inhibition) of about 0.5 nM to about 100 xcexcM. The preferred compounds show values of 0.5 nM to 100 nM.
As mentioned earlier, medicaments containing a compound of formula I are also an object of the present invention as is a process for the manufacture of such medicaments, which process comprises bringing one or more compounds of formula I and, if desired, one or more other therapeutically valuable substances into a galenical administration form.
The pharmaceutical compositions may be administered orally, for example in the form of tablets, coated tablets, dragees, hard or soft gelatin capsules, solutions, emulsions or suspensions. Administration can also be carried out rectally, for example using suppositories; locally or percutaneously, for example using ointments, creams, gels or solutions; or parenterally, for example using injectable solutions.
For the preparation of tablets, coated tablets, dragees or hard gelatin capsules the compounds of the present invention may be admixed with pharmaceutically inert, inorganic or organic excipients. Examples of suitable excipients for tablets, dragees or hard gelatin capsules include lactose, maize starch or derivatives thereof, talc or stearic acid or salts thereof.
Suitable excipients for use with soft gelatin capsules include for example vegetable oils, waxes, fats, semi-solid or liquid polyols etc.; according to the nature of the active ingredients it may however be the case that no excipient is needed at all for soft gelatin capsules.
For the preparation of solutions and syrups, excipients which may be used include for example water, polyols, saccharose, invert sugar and glucose. For injectable solutions, excipients which may be used include for example water, alcohols, polyols, glycerin, and vegetable oils. For suppositories, and local or percutaneous application, excipients which may be used include for example natural or hardened oils, waxes, fats and semi-solid or liquid polyols.
The pharmaceutical compositions may also contain preserving agents, antioxidants, solubilising agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts for the variation of osmotic pressure, buffers, coating agents or antioxidants. They may also contain other therapeutically valuable agents.
The dosages in which the compounds of formula I are administered in effective amounts depend on the nature of the specific active ingredient, the age and the requirements of the patient and the mode of application. In general, dosages of 0.1-100 mg/kg body weight per day come into consideration, although the upper limit quoted can be exceeded when this is shown to be indicated.
The following specific examples are provided as a guide to assist in the practice of the invention, and are not intended as a limitation on the scope of the invention.